RESUMO
Objective: Dual inhibition of mitogen-activated protein kinase [MAPK] kinase [also known as MEK] and transforming growth factor beta [TGFbeta] type I receptors by PD0325901 and SB431542, known as R2i has been introduced as a highly efficient approach to the generation of mouse embryonic stem cells [ESC]. In the present study, we investigated the molecular mechanisms underlying ESC derivation in the R2i condition
Materials and Methods: In this experimental study, zona-free whole E3.5 blastocysts were seeded on mouse embryonic fibroblast [MEF] feeder cells in both R2i and serum conventional media. The isolated inner cell mass [ICM], ESCs and the ICM-outgrowths were collected on days 3, 5 and 7 post-blastocyst culture for quantitative real timepolymerase chain reaction [qRT-PCR] analysis as well as to assess the DNA methylation status at the time points during the transition from ICM to ESC
Results: qRT-PCR revealed a significantly higher expression of the pluripotency-related genes [Oct4, Nanog, Sox2, Rex1, Dppa3, Tcf3, Utf1, Nodal, Dax1, Sall4 and beta-Catenin] and lower expression of early differentiation genes [Gata6, Lefty2 and Cdx2] in R2i condition compared to the serum condition. Moreover, the upstream region of Oct4 and Nanog showed a progressive increase in methylation levels in the upstream regions of the genes following in R2i or serum conditions, followed by a decrease of DNA methylation in ESCs obtained under R2i. However, the methylation level of ICM outgrowths in the serum condition was much higher than R2i, at levels that could have a repressive effect and therefore explain the absence of expression of these two genes in the serum condition
Conclusion: Our investigation revealed that generation of ESCs in the ground-state of pluripotency could be achieved by inhibiting the MEK and TGF-beta signaling pathways in the first 5 days of ESC derivation